Immunoregulatory abnormalities have been shown to exist in a wide variety of "autoimmune" and chronic inflammatory diseases, including systemic lupus erythematosis, chronic rheumatoid arthritis, type I and II diabetes mellitus, inflammatory bowel disease, biliary cirrhosis, uveitis, multiple sclerosis and other disorders such as Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, psoriasis, ichthyosis, and Graves ophthalmopathy. Although the underlying pathogenesis of each of these conditions may be quite different, they have in common the appearance of a variety of autoantibodies and self-reactive lymphocytes. Such self-reactivity may be due, in part, to a loss of the homeostatic controls under which the normal immune system operates.
Similarly, following a bone-marrow or an organ transplantation, the host lymphocytes recognize the foreign tissue antigens and begin to produce antibodies which lead to graft rejection.
One end result of an autoimmune or a rejection process is tissue destruction caused by inflammatory cells and the mediators they release. Anti-inflammatory agents such as NSAID's and corticosteroids act principally by blocking the effect or secretion of these mediators but do nothing to modify the immunologic basis of the disease. On the other hand, cytotoxic agents, such as cyclophosphamide, act in such a nonspecific fashion that both the normal and autoimmune responses are shut off. Indeed, patients treated with such nonspecific immunosuppressive agents are as likely to succumb from infection as they are from their autoimmune disease.
Cyclosporin A, which was approved by the US FDA in 1983, is currently the leading drug used to prevent rejection of transplanted organs. The drug acts by inhibiting the body's immune system from mobilizing its vast arsenal of natural protecting agents to reject the transplant's foreign protein. Though cyclosporin A is effective in fighting transplant rejection, it is nephrotoxic and is known to cause several undesirable side effects including kidney failure, abnormal liver function and gastrointestinal discomfort.
Newer, safer drugs exhibiting fewer side effects are constantly being searched for in the field. The present invention provides for an immunosuppressant peptide that is a selective inhibitor of a voltage dependent potassium channel, K.sub.v 1.3, that is found on human T-lymphocytes.
Potassium channels modulate a number of cellular events such as muscle contraction, neuro-endocrine secretion, frequency and duration of action potentials, electrolyte homeostasis, and resting membrane potential. These channels comprise a family of proteins that have been classified according to their biophysical and pharmacological characteristics. Inhibition of K.sup.+ channels, in their role as modulators of the plasma membrane potential in human T-lymphocytes, has been postulated to play a role in eliciting immunosuppressive responses. In regulating membrane potential, K.sup.+ channels play a role in the regulation of intracellular Ca.sup.++ homeostasis, which has been found to be important in T-cell activation. The biochemical characterization of K.sup.+ channels is underdeveloped, due to the paucity of selective high affinity probes.
Functional voltage-gated K.sup.+ channels can exist as multimeric structures formed by the association of either identical or dissimilar subunits. This phenomena is thought to account for the wide diversity of K.sup.+ channels found in different tissues. Despite the rapid advances in the molecular biology of K.sup.+ channels, subunit compositions of native K.sup.+ channels and the physiologic role that particular channels play are, in most cases, still unclear. To address these problems, potent, selective probes for channels of interest must be identified.
The K.sub.v 1.3 channel is a voltage-gated potassium channel that is found in neurons, blood cells, osteoclasts and T-lymphocytes. The Chandy and Cahalan laboratories proposed a hypothesis that blocking the K.sub.v 1.3 channel would illicit an immunosuppressant response. (Chandy et al., J. Exp. Med. 160, 369, 1984; Decoursey et al., Nature, 307, 465, 1984). However, the K.sup.+ channel blockers employed in their studies were non-selective. Until the present invention, no specific inhibitor of the K.sub.v 1.3 channel existed to test this hypothesis. Although a laboratory (Price et al., Proc. Natl. Acad. Sci. USA, 86, 10171, 1989) showed that charybdotoxin would block K.sub.v 1.3 in human T cells, charybdotoxin was subsequently shown to inhibit four different K.sup.+ channels (K.sub.v 1.3 and three distinct small conductance Ca.sup.2+ activated K.sup.+ channels) in human T lymphocytes, limiting the use of this toxin as a probe for the physiological role of K.sub.v 1.3 (Leonard et al., Proc. Natl. Acad. Sci. USA, 89, 10094, 1992). Since MgTX is a selective K.sub.v 1.3 inhibitor, it is useful to show that blocking of K.sub.v 1.3 will inhibit T cell activation (Lin et al., J. Exp. Med, 177, 637, 1993).
Scorpion venoms have been recognized as a source of peptidyl inhibitors of various types of K.sup.+ channels. Some of these peptides have been purified to homogeneity and their properties characterized. A preliminary report describing the presence of a specific peptidyl voltage-gated potassium channel inhibitor in C. margaritatus venom that is homologous with NxTX has been made in abstract form (Novick, et al. (1991) Biophys J 59, 78). The most extensively studied of these toxins is charybdotoxin (ChTX). See U.S. Pat. No. 4,906,867. ChTX is a thirty-seven amino acid peptide isolated from venom of the old world scorpion Leiurus quinquestriatus var. hebraeus. Originally described as an inhibitor of the high-conductance, Ca.sup.2+ -activated K.sup.+ (Maxi-K) channel present in muscle and neuro-endocrine cells, ChTX was later found also to inhibit a number of different medium- and small-conductance Ca.sup.2+ -activated K.sup.+ channels, as well as a voltage-dependent K.sup.+ channel (K.sub.v 1.3) In each case, channel inhibition occurs with similar potency, in the low nanomolar range. Therefore, caution has to be exercised when using ChTX to study the physiological role of a given channel in a tissue of interest. A related toxin, iberiotoxin (IbTX), shares 68% sequence homology with ChTX and selectively blocks the Maxi-K channel. Other peptidyl inhibitors, such as limbatustoxin (LbTX) and kaliotoxin (KTX), have also been shown to possess greater selectivity for the Maxi-K channel. Other peptidyl toxins homologous to ChTX have been identified (e.g., noxiustoxin.). Noxiustoxin is a high-affinity blocker of K.sub.v 1.3, but because it also inhibits the delayed rectifier K.sup.+ channel of squid axon and the Maxi-K channel from skeletal muscle, and is not selective for K.sub.v 1.3, it is not a satisfactory probe of K.sub.v 1.3 channels.
The peptide of the present invention, MgTX, represents a unique tool with which to probe the function of K.sub.v 1.3. This channel has been identified as the major voltage-dependent K.sup.+ conductance in peripheral human T-lymphocytes. Since human T-lymphocytes contain, in addition to K.sub.v 1.3, several distinct small-conductance Ca.sup.2+ -activated K.sup.+ channels that are also blocked by ChTX, ChTX is inadequate for assigning the role of any specific channel in the control of T cell proliferation. MgTX has recently been demonstrated to depolarize human T cells (Leonard et al., Proc. Natl. Acad. Sci. U.S.A. 89, 10094, 1992) and to prevent activation and proliferation of these cells mediated by Ca.sup.2+ -dependent pathways (Lin et al., J. Exp. Med., 177, 637, 1993).
Venom of the new world scorpion Centruroides margaritatus was determined to contain an activity selectively directed against voltage-dependent K.sup.+ channels: it inhibited binding of [.sup.125 I]ChTX to K.sub.v 1.3 channels in rat brain synaptosomal membranes, but not to Maxi-K channels in smooth muscle sarcolemma. This invention comprises the purification, from this venom, of margatoxin (MgTX), its primary sequence and characterization as a K.sub.v 1.3 inhibitor, and the expression of recombinant MgTX in E. coli. MgTX is structurally related to other known K.sup.+ channel blocking peptides, but is distinguished by its potent and selective blockade of K.sub.v 1.3. Given these properties, MgTX represents a useful tool for studying the physiologic role of K.sub.v 1.3. This invention also relates to the construction of a gene encoding MgTX and the expression of this gene in E. coli to produce recombinant MgTX.